Coupling of experimental and computational approaches for the development of new dendrimeric nanocarriers for gene therapy.

2013/2014Gene therapy is increasingly critical in the treatment of different types of maladies. The approach of gene therapy can be fundamental in dealing with many kinds of tumors, viral infections (e.g., HIV, HSV), and disturbs linked to genetic anomalies. However, the use of nucleic acids is limited by their ability to reach their action site—the target cell and, often, the inside of its nucleus. Dendrimers, on the other hand, are an interesting kind of polymers, the general synthetic scheme of which is relatively of recent development (∼1980). Among the many possible uses of these polymers, they revealed themselves as great nanocarriers for drugs in general, and particularly for genetic material. Many of the properties of these molecules are directly linked to their structure, and this in turn is critically influenced by their molecular composition. Exploiting in silico techniques, we can reveal many informations about the atomistic structure of dendrimers, some of which are otherwise difficult to gather. The interactions between the carrier and its cargo, and also with all the biological systems that are interposed between the administration and the reaching of the target (e.g., serum proteins, lipid membranes. . . ) are of critical importance in the development of new dendrimers for gene therapy. These interactions can be described and studied at a detail once unthinkable, thanks to the in silico simulation of these systems. In this thesis many different molecular simulation techniques will be employed to give a characterization as precise as possible of the structure and interactions of new families of dendrimers. In particular two new families of dendrimers (viologen and carbosilane) will be structurally characterized, and their interactions with albumin and two oligodeoxynucleotide, respectively, will be described. Then, the point of view of these interactions will be changed: the interactions between a fifth generation triethanolamine-core poly(amidoamine) dendrimer (G5 TEA-core PAMAM) and a sticky siRNA will be studied, varying the length and chemical compositions of the overhangs of the siRNA. Studying dendrimers the use of new molecular simulations techniques were deepened, and such techniques will be employed in other parallel projects. We’ll see the steered molecular dynamic method applied in the study of one mutation of the SMO receptor. The development of biological membranes models (that will be used in future to study the interactions of dendrimers with such membranes) was also used to refine and better characterize the σ1 receptor 3D model, previously developed by our research group. A detailed characterization of the putative binding site of this receptor will be given, employing this refined model.La terapia genica si sta rivelando sempre più importante nel trattamento di diversi tipi di malattie. Da diversi tipi di tumori alle infezioni virali, quale ad esempio da HIV, fino anche a malattie legate ad anomalie genetiche sono tutti disturbi in cui l’approccio della terapia genica può rivelarsi fondamentale. L’utilizzo di acidi nucleici quali agenti terapeutici è fortemente limitato dalla possibilità di portare queste molecole al loro sito d’azione—la cellula bersaglio e, spesso, l’interno del nucleo di quest’ultima. I dendrimeri d’altro canto sono un interessante tipo di polimero, di cui lo schema generale di sintesi è relativamente recente (∼1980). Tra i diversi loro utilizzi, questi polimeri si sono rivelati anche ottimi agenti di trasporto per farmaci, ed in particolare per materiale genetico. Molte delle proprietà di queste molecole derivano direttamente dalla loro struttura, e questa è influenzata criticamente dalla loro composizione. Mediante tecniche in silico è possibile avere molte informazioni riguardo la struttura dei dendrimeri, alcune delle quali sono altrimenti difficilmente ottenibili. L’interazione tra il trasportatore ed il suo “carico”, come anche con tutti i diversi sistemi biologici che si frappongono tra la somministrazione ed il raggiungimento dell’obbiettivo (ad es. proteine seriche, membrane lipidiche...) è un parametro chiave nello sviluppo di nuovi dendrimeri per la terapia genica. Queste interazioni possono essere descritte e studiate con un dettaglio un tempo impossibile, mediante la simulazione in silico di tali sistemi. In questo lavoro di tesi diverse tecniche di simulazione molecolare saranno utilizzate al fine di dare una caratterizzazione quanto più precisa possibile della struttura e delle intera- zioni di nuove classi di dendrimeri. In particolare sarà data una descrizione strutturale di due nuove famiglie di dendrimeri viologeni e carbosilani, e delle loro interazioni rispettivamente con albumina e due diversi oligodeossinucleotidi. Si alternerà poi il punto di vista da cui studiare tale interazione: sarà data una descrizione dell’interazione tra un dendrimero po- liammidoamminico a nucleo trietanolamminico (TEA-core PAMAM) di generazione 5 e uno sticky siRNA, al variare della lunghezza e tipo di “braccia” del siRNA. Nello studio di dendrimeri alcune nuove tecniche di simulazione molecolare sono state approfondite, e tali tecniche sono state utilizzate anche in altri progetti paralleli. Vedremo la steered molecular dynamic applicata allo studio di una mutazione del recettore SMO. Lo sviluppo di modelli di membrane biologiche (utile in futuro per lo studio dell’interazione di dendrimeri con tali membrane) è stato utilizzato per perfezionare e meglio caratterizzare il modello tridimensionale del recettore σ1, precedentemente sviluppato dal nostro gruppo di ricerca. Una caratterizzazione dettagliata del sito di binding putativo di questo recettore sarà descritta, usando tale perfezionato modello.XXVII Ciclo198

Molecular Features for Probing Small Amphiphilic Molecules with Self-Assembled Monolayer Protected Nanoparticles

The sensing of small molecules poses the challenge to develop devices able to discriminate between compounds that may be structurally very similar. Here, attention has been paid to the use of self-assembled monolayer (SAM)-protected gold nanoparticles since they enable a modular approach to tune single-molecule affinity and selectivity simply by changing functional moieties (i.e. covering ligands), alongside with multivalent molecular recognition. To date, the discovery of monolayers suitable for a specific molecular target relies on trial-and-error approaches, with ligand chemistry being the main criteria used to modulate selectivity and sensitivity. By using molecular dynamics, we showcase that either individual molecular characteristics and/or collective features such as ligand flexibility, monolayer organization, ligand local ordering, and interfacial solvent properties can also be exploited conveniently. The knowledge of the molecular mechanisms that drive the recognition of small molecules on SAM covered nanoparticles will critically expand our ability to manipulate and control such supramolecular systems

Evolution from Covalent to Self-Assembled PAMAM-Based Dendrimers as Nanovectors for siRNA Delivery in Cancer by Coupled in Silico-Experimental Studies. Part II: Self-Assembled siRNA Nanocarriers

In part I of this review, the authors showed how poly(amidoamine) (PAMAM)-based dendrimers can be considered as promising delivering platforms for siRNA therapeutics. This is by virtue of their precise and unique multivalent molecular architecture, characterized by uniform branching units and a plethora of surface groups amenable to effective siRNA binding and delivery to e.g., cancer cells. However, the successful clinical translation of dendrimer-based nanovectors requires considerable amounts of good manufacturing practice (GMP) compounds in order to conform to the guidelines recommended by the relevant authorizing agencies. Large-scale GMP-standard high-generation dendrimer production is technically very challenging. Therefore, in this second part of the review, the authors present the development of PAMAM-based amphiphilic dendrons, that are able to auto-organize themselves into nanosized micelles which ultimately outperform their covalent dendrimer counterparts in in vitro and in vivo gene silencing

Perceptions and Misconceptions in Molecular Recognition: Key Factors in Self-Assembling Multivalent (SAMul) Ligands/Polyanions Selectivity

Biology is dominated by polyanions (cell membranes, nucleic acids, and polysaccharides just to name a few), and achieving selective recognition between biological polyanions and synthetic systems currently constitutes a major challenge in many biomedical applications, nanovectors-assisted gene delivery being a prime example. This review work summarizes some of our recent efforts in this field; in particular, by using a combined experimental/computation approach, we investigated in detail some critical aspects in self-assembled nanomicelles and two major polyanions-DNA and heparin

Evolution from Covalent to Self-Assembled PAMAM-Based Dendrimers as Nanovectors for siRNA Delivery in Cancer by Coupled In Silico-Experimental Studies. Part I: Covalent siRNA Nanocarriers

Small interfering RNAs (siRNAs) represent a new approach towards the inhibition of gene expression; as such, they have rapidly emerged as promising therapeutics for a plethora of important human pathologies including cancer, cardiovascular diseases, and other disorders of a genetic etiology. However, the clinical translation of RNA interference (RNAi) requires safe and efficient vectors for siRNA delivery into cells. Dendrimers are attractive nanovectors to serve this purpose, as they present a unique, well-defined architecture and exhibit cooperative and multivalent effects at the nanoscale. This short review presents a brief introduction to RNAi-based therapeutics, the advantages offered by dendrimers as siRNA nanocarriers, and the remarkable results we achieved with bio-inspired, structurally flexible covalent dendrimers. In the companion paper, we next report our recent efforts in designing, characterizing and testing a series of self-assembled amphiphilic dendrimers and their related structural alterations to achieve unprecedented efficient siRNA delivery both in vitro and in vivo

Mixed-monolayer functionalized gold nanoparticles for cancer treatment: Atomistic molecular dynamics simulations study.

Gold nanoparticles (AuNPs) are employed as drug carriers due to their inertness, non-toxicity, and ease of synthesis. An experimental search for the optimal AuNP design would require a systematic variation of physico-chemical properties which is time-consuming and expensive. Computational methods provide quicker and cheaper approach to complement experiments and provide useful guidelines. In this paper, we performed atomistic molecular dynamics simulations to study how the size, hydrophobicity, and concentration of the drug affect the structure of functionalized AuNPs in the aqueous environment. We simulated two groups of nano-systems functionalized with a zwitterionic background ligand, and a ligand carrying a drug (Quinolinol or Panobinostat). Results indicate that in the case of a hydrophobic drug (Quinolinol), the hydrophobicity drives the conformation changes of the coating layer. The tendency of the hydrophobic drug to reduce its solvent-accessible surface results in a decrease of the coating thickness and the overall NP size. Although the amount of accessible drug can be increased by increasing its initial concentration, it will compromise the solubility of the system. In the case of a hydrophilic drug (Panobinostat), the ligand in excess has a dominant influence on the final structure of the coating conformations. The percentage of accessible drug is significantly higher than in the hydrophobic systems for any given ratio. It implies that for hydrophilic systems we can generally expect higher biological efficiency. Our results highlight the importance of taking into account physico-chemical properties of drugs and ligands when developing gold-based nanosystems, especially in the case of hydrophobic drugs

Probing multiscale factors affecting the reactivity of nanoparticle-bound molecules

I. K. M., W. E., E. J. H, S. S. and E. R. K. are grateful for funding from the Leverhulme Trust [RPG-2015-042], the Engineering and Physical Sciences Research Council [EP/K016342/1], the University of St Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) [Ph.D. studentship to SS: EP/L016419/1]. D. M. and P.P thank the Italian Ministry of University Research (MIUR) for funding [RBSI14PBC6].The structures and physicochemical properties of surface-stabilizing molecules play a critical role in defining the properties, interactions, and functionality of hybrid nanomaterials such as monolayer-stabilized nanoparticles. Concurrently, the distinct surface-bound interfacial environment imposes very specific conditions on molecular reactivity and behavior in this setting. Our ability to probe hybrid nanoscale systems experimentally remains limited, yet understanding the consequences of surface confinement on molecular reactivity is crucial for enabling predictive nanoparticle synthon approaches for postsynthesis engineering of nanoparticle surface chemistry and construction of devices and materials from nanoparticle components. Here, we have undertaken an integrated experimental and computational study of the reaction kinetics for nanoparticle-bound hydrazones, which provide a prototypical platform for understanding chemical reactivity in a nanoconfined setting. Systematic variation of just one molecular-scale structural parameter—the distance between reactive site and nanoparticle surface—showed that the surface-bound reactivity is influenced by multiscale effects. Nanoparticle-bound reactions were tracked in situ using 19F NMR spectroscopy, allowing direct comparison to the reactions of analogous substrates in bulk solution. The surface-confined reactions proceed more slowly than their solution-phase counterparts, and kinetic inhibition becomes more significant for reactive sites positioned closer to the nanoparticle surface. Molecular dynamics simulations allowed us to identify distinct supramolecular architectures and unexpected dynamic features of the surface-bound molecules that underpin the experimentally observed trends in reactivity. This study allows us to draw general conclusions regarding interlinked structural and dynamical features across several length scales that influence interfacial reactivity in monolayer-confined environments.PostprintPeer reviewe

Patchy and Janus Nanoparticles by Self-Organization of Mixtures of Fluorinated and Hydrogenated Alkanethiolates on the Surface of a Gold Core

The spontaneous self-organization of dissimilar ligands on the surface of metal nanoparticles is a very appealing approach to obtain anisotropic "spherical". systems. In addition to differences in ligand length and end groups, a further thermodynamic driving force to control the self-assembled monolayer organization may become available if the ligands are inherently immiscible, as is the case of hydrogenated (H-) and fluorinated (F-) species. Here, we validate the viability of this approach by combining F-19 NMR experiments and multiscale molecular simulations on large sets of mixed-monolayer-protected gold nanoparticles (NPs). The phase segregation of blends of F- and H-thiolates grafted on the surface of gold NPs allows a straightforward approach to patterned mixed monolayers, with the shapes of the monolayer domains being encoded in the structure of the F/H-thiolate ligands. The results obtained from this comprehensive study offer molecular design rules to achieve a precise control of inorganic nanoparticles protected by specifically patterned monolayers

Mallard Blue binding to heparin, its SDS micelle-driven de-complexation, and interaction with human serum albumin : A combined experimental/modeling investigation

Heparin is a sulfated glycan widely used as anticoagulant in medicine. Mallard Blue (MalB), a small cationic dye developed in our laboratories, is able to detect heparin in serum and plasma in a dose-response manner, with performance superior to its direct competitors. However, many aspects of MalB/heparin binding still remain to be explored which, once solved, may foster the clinical use of MalB. Among these, the characterization of the energetics that drives the MalB/heparin binding process, the competition for MalB binding by other polyanions (e.g., negatively-charged surfactant micelles), and the interaction of MalB with serum proteins are of particular interest. This work fills this gap by means of a combination of experimental investigations (UV-visible spectroscopy and isothermal titration calorimetry), and computational approaches based on molecular dynamics (MD) simulation techniques. In combination, the results obtained show that MalB efficiently binds to both heparin and SDS, with the binding being enthalpic in nature; yet, SDS is able to extract MalB from its complex with heparin when the surfactant is in its self-assembled form, the driving force underlying SDS-induced MalB/heparin de-complexation being entropic in nature as the two enthalpies of binding effectively cancel each other out. Once bound to SDS, the dye remains electrostatically bound to the micellar surface and does not penetrate the micelle palisade layer, as verified by steered molecular dynamics/umbrella sampling simulations. Finally, the affinity of MalB for human serum albumin (HSA), the most abundant plasma protein, is found to be lower than that for heparin, confirming the ability of the dye to work in complex physiological environments

Some things old, new and borrowed: Delivery of dabrafenib and vemurafenib to melanoma cells via self-assembled nanomicelles based on an amphiphilic dendrimer

Two clinically approved anticancer drugs targeting BRAF in melanoma patients - dabrafenib (DAB) and vemurafenib (VEM) - have been successfully encapsulated into nanomicelles formed upon self-assembly of an amphiphilic dendrimer AD based on two C18 aliphatic chains and a G2 PAMAM head. The process resulted in the formation of well-defined (∼10 nm) core-shell nanomicelles (NMs) with excellent encapsulation efficiency (∼70% for DAB and ∼60% for VEM) and good drug loading capacity (∼27% and ∼24% for DAB and VEM, respectively). Dynamic light scattering (DLS), transmission electron microscopy (TEM), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular simulation (MS) experiments were used, respectively, to determine the size and structure of the empty and drug-loaded nanomicelles (DLNMs), along with the interactions between the NMs and their cargoes. The in vitro release data revealed profiles governed by Fickian diffusion; moreover, for both anticancer molecules, an acidic environment (pH = 5.0) facilitated drug release with respect to physiological pH conditions (pH = 7.4). Finally, both DAB- and VEM-loaded NMs elicited enhanced response with respect to free drug treatments in 4 different melanoma cell lines